Improving the characteristics of mozzarella cheese manufactured in an unconventional way and fortified with nanosalt and studying the effect of freezing on it

Manufacture of nanosodium chloride particles (nanosalt)

In this method, salt (NaCl) is used in a cylindrical mill to prepare nanoparticle powder from the active material. A mechanical grinder supplied by the German company Ritsch was used and operated at 400 rpm for 12 min, with two to three pauses. The samples were collected in sterile, opaque bottles and stored at room temperature until testing.

Experimental design

Nanodried sodium chloride particles were used to prepare low-sodium cheese. The treatments consisted of three different concentrations of nanotable salt (NaCl) (0.5%, 1%, and 1.5%). The three treatments were tested for chemical composition on day zero, and at weeks 1, 2, and 3. The cheese was sensory evaluated for appearance (5 scores), bitterness (10 scores), cracking (10 scores), texture (20 scores), flavour (45 scores), and color (10 scores).

Cheese production

Mozzarella cheese was produced at the Dairy Processing Laboratory of the College of Agricultural Engineering Sciences, University of Baghdad. Cheese production, including coagulation, cutting, filtration, and curd formation, was conducted on a pilot scale under sterile conditions as previously described.²⁵

Samples of imitation mozzarella cheese were prepared as follows: cow milk was divided into five fractions. The first fraction was gradually acidified directly with 2% dilute lactic acid until a pH of 5.4 was reached and then refrigerated (T1). The second fraction was manufactured from cow milk according to the standard method described previously²⁶ which involves direct acidification with 2% dilute citric acid and freezing (T2) in a home freezer at −20°C for 21 days. The third, fourth, and fifth treatments (T3, T4, and T5) were produced according to the experimental design by adding 1% citric acid to fresh cow milk and salting it with nanosalt (0.5%, 1%, and 1.5%, respectively). All samples (T1) were cooked at 85°C for 10 min. Three replicates were performed for each treatment. Freshly produced cheese samples were analyzed to determine their chemical composition, physical and sensory properties

T1 = Comparison (control) treatment, Cooled treatment

T2 = Mozzarella cheese frozen treatment

T3 = Mozzarella cheese with 0.5% added nano salt

T4 = Mozzarella cheese with 1% added nano salt

T5 = Mozzarella cheese with 1.5% added nano salt

Sensory evaluation

Sensory tests were conducted on cheese samples subjected to different treatments at 0, 7, 14, and 21 days of storage, depending on the number of experienced evaluators (5) at the College of Agricultural Engineering Sciences, Department of Food Sciences, University of Baghdad. Sensory tests include grading the qualities of color, flavour, texture, opening, bitterness and external appearance, with a ranges of 10, 45, 20, 10, 10 and 5 degrees, respectively, according to the methods of Sameen et al.²⁹ and Abu-Foul (1990).³⁰

ISO 8589:2007 provides general guidelines for the design of test rooms intended for the sensory evaluation of products and requires criteria for plate size and training procedures as part of the overall methodology of the test. These general guidelines include requirements for the test area, preparation area, and office, with the caveat that the standard does not pertain to any specific product or type of testing.

Test Room Design: ISO 8589:2007 describes the basic or desired design requirements for a test room, which typically includes a testing area, preparation area, and desk.

Plate Size Standards: Standard sets of standards for determining the size of the plate used in the sensory evaluation. Importantly, the usual method gives wide recommendations in preference to precise measurements.

Training procedures

Although the specifications do not cover specialized training in detail, well known techniques include training processes for testers; however, the specifics of those processes can also vary on the basis of the product and type of test.

Application: These guidelines follow to the layout of test rooms for preferred sensory evaluation and do no longer cover testing facilities dedicated to specialized inspections or quality control within a factory.

Scanning electron microscopy

The samples were examined via S-4000T FESEM, and an analysis sample was applied to adhesive tabs on aluminum for SEM. The stubs were coated with platinum using an EMS 550X sputter coater (Electron Microscopy Sciences, Hatfield, PA). A JSM-6610 scanning electron microscope (Jeol Ltd., Tokyo, Japan) operating at an acceleration voltage of 15 kV and a vacuum of 0.00001 MPa was used to examine the materials and capture photographs.³¹

Loss of weight

The weight loss of the Mozzarella cheese slices was determined via Equation (1), where W₀ represents the original sample weight (day 0) and W_t denotes the sample weight at 21 days of storage.³²

Statistical analysis

The Statistical Analysis System-SAS (2018) program was used to detect the effects of the different treatments on the study parameters. Least significant difference (LSD) tests were used to compare the means in this study.³³

Protein content material (%) levels

Table 1 illustrates the protein content (percentage) changes within mozzarella cheese under different storage conditions, which include refrigeration (T1), freezing (T2), and addition of nanosalt at varying concentrations (0.5% = T3, 1% = T4, 1.5% = T5), and the samples were stored at 5° C for a period of 21 days. Based on the interpretation of the data, it is established that there was a significant consistency in the protein percentages across the days 0 and 21, and the minimum and statistically nonsignificant (NS) changes were consistent with the least significant difference (L.S.D = 1.07), therefore indicating that the protein content was relatively stable during the entire length of the store period. At the treatment stage, T4 (1% nanosalt) exhibited the maximum elevated protein content (25.90%), in contrast to the results of the T1 treatment (25.46%), suggesting that the application of nanosalt at a moderate concentration may promote the stability of protein construction in cheese by mitigating water loss and inhibiting enzymatic reactions that contribute to the degradation of proteins. On the other hand, a lower percentage was observed on day 0 in T3 (25.00%), which can be attributed to a minor difference in protein distribution at the beginning of storage.

Levels of fat content (%)

The values in Table 1 indicate that the fat percentage changes between days 0 and 21 were slight across all the treatments, with no significant differences recorded according to the LSD value (1.78 NS), indicating stable fat content across all the treatments. In the T1 treatments, fat (%) increased from 20.69% to 20.80% after 21 days, a nonsignificant increase that is likely attributed to a slight loss of moisture during storage, which increases the relative fat concentration. An identical pattern become determined in T2, where the fat percentage remained nearly constant (20.50% to 20.58%), indicating that freezing did no longer had a negative an effect on fat stability for the duration of this time. In the nanosalt-treated samples, slightly decreased lipid contents were recorded on day zero, specifically at T3 (19.30%), but increased to 19.75% over time, suggesting that the nanosalt helped limit lipid loss for the duration of storage, likely by stabilizing the cellular membrane or lowering the enzyme activity that influences the lipid balance. In the T4 treatment, the fat content remained approximately regular (19.98% to 19.88%), indicating that this specific concentration can be optimal for preserving cheese additives without changing their residences.

Levels of Ash content (%)

The results in Table 1 show substantial variations in ash content (P ≤ 0.05) among the various storage treatments. These effects indicate that the addition of nanosalt notably affected the mineral content of cheese, with T3 and T5 resulting in the highest ash content after 21 days in comparison to the other treatments. In treatment T5 (1.5% nanosalt), the ash content increased from 3.29% on day zero to 3.71% on day 21, reflecting the accumulation of mineral salts within the cheese due to the better concentration of salt introduced.

Levels of Acidity (%)

The final outcome showed that there was a minimal, nonsignificant (NS) difference in the level of acidity among the various treatments over time (21 days) of storage at 5°C as reflected by the LSD value (0.167). A slight increase in acidity was reported in the control treatment (T1) (0.55% 0.62%)and the freezing treatment (T2). This growth is explained by natural bacterial processes that take place during storage and produce organic acids due to the breakdown of lactose.⁸

pH values

The results in the table showed slight changes in pH values ​​over the storage period (21 days at 5°C) between the different treatments, with no significant differences (L.S.D = 0.402, NS). In contrast, the nanosalt treatments (T3, T4, and T5) resulted in wed relatively stable pH values, with T4 even slightly increasing from 5.32 to 5.35.

Sensory attributes are vitally important and directly affect the consumer acceptance and market success of cheese,⁵⁴ Table 2 shows the results of the sensory evaluation of mozzarella cheese treated with nanosalt during a refrigerated storage period of 21 days at 5°C, according to the following characteristics: external appearance, bitter taste (bitterness), holes, texture, flavour, and color. The results revealed significant differences (P ≤ 0.05) in bitterness, texture, and flavour only, whereas no significant differences were recorded in external appearance, holes, or color according to the LSD value.

The external appearance, opening, and color parameters maintained full or near full evaluation scores (5/5 and 10/10), indicating good morphological stability and no effect on the optical properties during storage, even during the nanosalt treatments. This finding is in line with studies that have indicated that nanoprocessing techniques do not cause noticeable changes in the morphological properties of cheese.

For bitterness, significant differences were observed between treatments, with T1 and T2 showing lower scores after 14 and 21 days (e.g., 7/10 in T1 at 21 days). In contrast, treatments T3–T5 (nanosalt) maintained decreased bitterness ranges and higher ratings;

For Textures, remedies T2, T3, T4, and T5 confirmed the right consistency in texture (20/20 or close to it), whereas T1 recorded a gradual decline after 14 and 21 days which were recorded (17/20, and 15/20) respectively.

The flavour consequences shown in Table 2 show large variations (LSD = 3.87) at P ≤ 0.05* among the exceptional treatments over 21 days of storage at 5°C. The T1 group started with an excellent flavour rating (45/45) on day zero; however, a clear decline over time was recorded, reaching 38 on days 14 and 35 on day 21. In T2, despite the decrease become much less excessive, the taste rankings reduced from 45, 41, 43 and 41, respectively, to zero, 7, 14, and 21, indicating that freezing reduces the price of decay in comparison with refrigeration; however, freezing does not completely prevent flavour loss. T3 (0.5 % nanosalt) showed the greatest flavour stability, maintaining the full score (45/45) untill day 14, and decreasing the most effective score to 43 via day 21, suggesting that the nanosalt performs a role in reducing microbial hobbies or oxidation, which could affect taste. The T4 treatment (1% nanosalt) resulted in a proper taste balance in the first week (45 → 40 on day 7); however, the balance stabilized at forty until day 21. These findings suggest that increasing the salt concentration above 0.5% did not result in further improvement, but may have a slight adverse effect on the flavour balance. The T5 treatment (1.5% nanosalt) recorded lower flavour scores which were recorded 40/45 after 7, and 14 days, Sample T5 was damaged, so no values ​​were recorded at 21 days of storage ( Table 2).

The results of the weight loss data in Table 3 for mozzarella cheese indicate significant differences between the different treatments during refrigerated storage at 5°C for 21 days, indicating that the processing method and type of additive clearly affect the rate of weight loss during storage. The control treatment (T1) showed a slight weight reduction from 57.69 g to 56.97 g,

In the T2 (freezing) treatment, a surprising weight increase was observed, from 59.90 grams to 60.80 grams. This can be due to external moisture absorption all through thawing or a mild weight variant resulting from freezing and inner moisture redistribution. The results of the evaluation revealed that remedy T3 (0.5% nanosalt) had excellent weight stability, with the burden decreasing from 28.72 grams to the handiest 26.95 grams.

Conversely, treatments T4 and T5 (1% and 1.5 % nanosalt) confirmed more weight loss than did the other treatments, regardless of the addition of the same additive. The weight reduction in T4 was approximately 5.94 grams, whereas that in T5 was approximately 7.00 grams.

Protein content material (%) levels

The established stability of the protein content can be attributed to the structural nature of the cheese proteins which are inherently strong, and the lack of suitable enzymes or microbial activity which can break down the proteins in this relatively limited time.⁴²

Recent studies have shown the beneficial effects of nanosalt treatment stabilizing milk components, particularly proteins through interactions with the surface charge sites of protein molecules leading to decreased proteolysis and changes in the tertiary structure of the protein.⁴³ Furthermore, mild refrigeration conditions also slow endogenous or microbial protease activity, leading to the preservation of protein disulfide without much degradation.⁴⁴

Levels of fat content (%)

The lipid content ratio during the storage period reflects the efficacy of refrigeration storage conditions in maintaining the structural pattern of cheese and reducing the amount of microbial or enzyme activity which may lead to lipolysis. Recent studies have that nanocomposites, composed of nanosalt, contribute to preventing the attack on lipids caused by oxidation or loss via improved distribution of salt and improved the ability of the protein community to entrap fats.⁴⁵ Additionally, Lauculien et al.⁴⁶ reported that the slight change in fat content was not due to fat loss, but due to water loss, namely, the usa of unfastened packaging or water activity changed.

Levels of Ash content (%)

The increased ash content may be attributed to the specific properties of nanosalt, mainly its small size and high specific surface area, which promote its interaction with proteins and other cheese components. This characteristic helps to incorporate it into the microstructure of cheese and enhance its mineral retention and increase its ash content with increasing concentration.

Levels of Acidity (%)

The samples treated with nanosalt (T3-T5) were more stable with respect to acidity levels with only a minimal decrease in T3, 0.60 to 0.53 %. This is an indication of the role of nanosalt in inhibiting microbial activity and acid production during storage,⁴ which helps to enhance the stability of cheese and sensory protection. Overall, these findings indicate that the application of nanosalt can potentially prevent the negative influence on acidic stability without a harmful impact on the quality of cheeses.

pH values

The chemical interactions among the essential structural components of cheese depend on the pH. Consequently, pH directly affects the structural and rheological characteristics of cheese.⁴⁵ A slight decrease in pH was detected in both the first (T1) and the second (T2) treatments. This is because calcium phosphate plays a vital role in determining the curdling properties of cheese, which is enhanced by a reduction in the pH from 6.0 to 5.1.⁴⁷ This in turn led to a higher level of lactate in the protein mixture and a greater level of calcium phosphate as observed in the gelling process thus resulting in a lower pH. Furthermore, the phosphate anion neutralizes the hydrogen ions (H+) and the phosphate anion concentration during the micellization of calcium phosphate increases with cooling, which may cause a reduction in the pH, as the phosphate anion forms organic acids.⁴⁸ In contrast, the nanosalt treatments (T3, T4, and T5) resulted in relatively stable pH values, indicating that nanosalt has a larger surface area and better distribution in the medium, which increases its effectiveness in inhibiting the growth of bacteria and yeasts, which typically causes changes in pH by producing organic acids (such as lactic or acetic acids). This decreases microbial activity and provides a stable pH.⁴⁹ A stable pH is a positive indicator of the quality and safety of product storage and also that t improves the sensory and physical attributes of the product during storage. The findings of this research are in line with the findings of Yazici et al.,⁵⁰ who reported no statistically significant interaction effect (P > 0.05) between the source of milk and the procedure of acidifying cheese on the pH of mozzarella cheese. The pH of the cheese was not affected by the source of milk as shown by other studies. Sameen et al.²⁹ found that the pH of mozzarella prepared from buffalo and cow milk was not greatly altered. This is in accordance to the findings of Emam and Nasir.⁵¹ Youssef et al.⁵² indicated that a decrease in cheese pH signifies the commencement of spoilage, probably because of carbon dioxide production through microorganisms as a consequence of lactate decomposition and amino acid decarboxylation on the cheese surface.⁵²

The discrepancies in the outcomes of the prevailing studies can be attributed to different manufacturing processes (i.e., differences in the dilution water to cheese ratio, salt concentration, and storage techniques), which may affect the percentage of buffering retailers (e.g. phosphates and proteins) to lactate.⁴⁸ The results of this study are consistent with those of a previous study.⁵³ Additional research is needed to investigate the factors influencing the pH of cured cheese.

For bitterness, this distinction is attributed to the precise bodily and chemical properties of nanosalt, which allow it to have an immediate or roundable effect on the enzymes and bacteria present in cheese. Nanosalt is thought to decrease the production of compounds associated with bitter flavour, which includes bitter peptides or brief-chain fatty acids, which are often produced by the use of the hobbies of proteolytic or lipolytic enzymes that are activated during storage. Furthermore, nanosalt debris can be labelled as bioporous nanocomposites, which might be organic polymeric substances that include various main levels: a nonstop phase, represented by a biopolymer shape, and a dispersed segment, composed of reinforcing materials with nanoscale sizes granging from 1–one hundred nanometres. This nanostructure endows the material with distinct and effective properties in food applications, consisting of structural flexibility, biocompatibility with food products, and biodegradability.⁵⁵

For Texture, this is attributed to the fact that nano salt facilitates strengthening the protein structure of cheese, lowering moisture loss and retaining its consistency. This is confirmed with the aid of current studies on the impact of nanosalts on the gel properties of dairy products. The findings of the cutting-edge approach align with those of Marshall (1991), who proposed that the versions are because of differing tiers of proteolysis, fat content, and physical and chemical characteristics of every cheese type.⁵⁶

This remedy has turned into a great sensory-clever. Some nanoparticles, including titanium dioxide (TiO2), which is applied as a bleaching agent and silicon dioxide (SiO2), which is used as an anticaking agent and flavour carrier, have been historically implemented in food products and are categorized as “generally recognized as safe” (GRAS) with the aid of the U.S. Food and Drug Administration. This aligns with the findings concerning the application of nanosalts as flavour preservatives.^57–59 The T4 and T5 treatment resulted in lower flavour scores, suggesting that high concentrations of nanosalt may negatively affect taste qualities due to excess saltiness or the interaction of salts with volatile components responsible for flavour.

The weight loss of mozzarella cheese, a natural loss, is likely attributed to water evaporation or leakage of some liquid cheese compounds over time. These effects are consistent with previous observations,⁶⁰ which indicated weight reduction after refrigeration and attributed this decrease to the thawing operation after frozen storage.

The consequences of remedy T3 (0.5% nanosalt) were excellent weight stability, indicating that this concentration of nanosalt efficiently reduced moisture loss, possibly by strengthening the protein structure of the cheese and stabilizing the water within its structure. This finding is consistent with what Aliabbasi & Emam-Djomeh (2024) mentioned, which indicated that the addition of nanosalts enhances the moisture retention capability and stabilizes the protein community of dairy merchandise.⁴

Treatments T4 and T5 (1% and 1.5 % nanosalt) confirmed more weight loss, possibly because high concentrations of nanosalt may have opposite effects, increasing the osmotic stress in the cheese and drawing water from the protein shape to stabilize it. This is supported by Salih et al. (2018), who reported that increased salt awareness in dairy products can decrease the ability of the product to keep water due to adjustments in the structural composition of the protein.¹⁴